Glucose-dependent GPER1 expression modulates tamoxifen-induced IGFBP-1 accumulation

Abstract

G protein-coupled estrogen receptor 1 (GPER1) is a seven-transmembrane receptor that mediates rapid cell signaling events stimulated by estrogens. While the role that GPER1 has in the modulation of E2-responsive tissues and cancers is well documented, the molecular mechanisms that regulate GPER1 expression are currently not well defined. The recently identified GPER1-dependent mechanism of tamoxifen action in breast cancer cells underscores the importance of identifying mechanisms that regulate GPER1 expression in this cell type. We hypothesized that GPER1 expression in breast cancer cells is sensitive to [D-glucose] and provide data showing increased GPER1 expression when cells were cultured in low [D-glucose]. To determine if the observed accumulation of GPER1 was AMP-activated protein kinase (AMPK)-dependent, small molecule stimulation or inhibition of AMPK was performed. AMPK inhibition decreased GPER1 accumulation in cells grown in low [D-glucose] while the AMPK-activating compound AICAR increased GPER1 accumulation in cells grown in high [D-glucose] media. Additionally, transfection of cells with a plasmid expressing constitutively active AMPK resulted in increased GPER1 accumulation. To determine if [D-glucose]-dependent GPER1 accumulation altered breast cancer cell response to tamoxifen, cells grown in the presence of decreasing [D-glucose] were co-treated with tamoxifen and IGFBP-1 transcription was measured. The results from these experiments reveal that D-glucose deprivation increased GPER1-mediated and tamoxifen-induced IGFBP-1 transcription suggesting that [D-glucose] may increase breast cancer cell sensitivity to tamoxifen. Taken together, these results identify a previously unknown mechanism that regulates GPER1 expression that modifies one aspect tamoxifen action in breast cancer cells.

Keywords: IGFBP-1; breast cancer; estrogen receptors; metabolism.

Figure 1

GPER1 expression is elevated in breast cancer cells cultured in low D-glucose-conditions. Immunoblot analysis of GPER1 expression in (A), MCF-7 cells and (B), T-47D cells cultured for 24 h in media containing the indicated [D-glucose]. Results are the average of at least three independent experiments and GPER1 expression was normalized to β-actin. Error bars are the standard error of the mean and statistical significance (P < 0.05) is noted using *.

Figure 2

GPER1 and phospho-AMPKα (Thr 172) accumulates in breast cancer cells cultured in low D-glucose conditions. Immunoblot analysis of GPER1 and pAMPK (Thr 172) accumulation in (A), MCF-7 cells and (B), T-47D cells cultured for 24 h in media containing the indicated [D-glucose]. Results are the average of three independent experiments and GPER1 expression was normalized to β-actin. Error bars are the standard error of the mean and statistical significance (P < 0.05) is noted using *.

Figure 3

D-glucose deprivation did not significantly change GPER1 expression or pAMPK (Thr 172) accumulation in ELT-3 or ELT-6 cells. Immunoblot analysis of GPER1 and pAMPK (Thr 172) accumulation in (A) ELT-3 cells and (B) ELT-6 cells cultured for 24 h in media containing the indicated [D-glucose]. Results are the average of three independent experiments and GPER1 expression was normalized to β-actin. Error bars are the standard error of the mean.

Figure 4

Reduced GPER1 expression observed after AMPK inhibition in ELT-3 and ELT-6 cells. Immunoblot analysis of GPER1 and pAMPK (Thr 172) accumulation in (A) ELT-3 cells and (B) ELT-6 cells cultured for 24 h in media containing the indicated dose of dorsomorphin. Results are the average of three independent experiments and GPER1 expression was normalized to β-actin. Error bars are the standard error of the mean and statistical significance (P < 0.05) is noted using *.

Figure 5

AMPK inhibition decreased GPER1 expression in MCF-7 cells cultured in low [D-glucose]. (A) Immunoblot analysis GPER1 expression and (B) quantitative real-time PCR analysis of GPER1 transcript level in MCF-7 cells cultured in low [D-glucose] after 24-h treatment with the indicated dose of dorsomorphin. Results are the average of 3 independent experiments. GPER1 protein expression was normalized using β-actin and GPER1 transcript levels were normalized using RPL30. Error bars are the standard error of the mean and statistical significance (P < 0.05) is noted using *.

Figure 6

AMPK activation induced GPER1 expression in breast cancer cells. Immunoblot analysis of GPER1 and pAMPK (Thr 172) accumulation in (A) MCF-7 cells and (B) T-47D cells cultured in 25 mM D-glucose and treated for 24 h with the indicated dose of AICAR. (C) Quantitative real-time PCR analysis of GPER1 transcript level in MCF-7 (left) and T-47D (right) cells cultured in 25 mM D-glucose and treated for 24 h with the indicated dose of AICAR. Quantitative results are the average of three independent experiments. GPER1 protein expression was normalized using β-actin and GPER1 transcript levels were normalized using RPL30. Error bars are the standard error of the mean and statistical significance (P < 0.05) is noted using *.

Figure 7

Expression of constitutively active AMPKα induced GPER1 expression in breast cancer cells. Immunoblot analysis of GPER1 expression 24 h post transfection with a plasmid expressing constitutively active AMPKα (aa1-312) in (A) MCF-7 cells and (B) T-47D cells. Results are the average of three independent experiments and GPER1 expression was normalized to β-actin. Error bars are the standard error of the mean and statistical significance (P < 0.05) is noted using *.

Figure 8

Low D-glucose concentration potentiates IGFBP-1 transcription in Tam-treated breast cancer cells. Quantitative real-time PCR analysis of IGFBP-1 transcript level in breast cancer cells cultured in serum-free DMEM with indicated [D-glucose] after 24-h treatment with (A) 1 µM Tam or (B) 1 µM Tam + 1 µM G-36. Results are the average of three independent experiments and IGFBP-1 transcript levels were normalized using RPL30. Error bars are the standard error of the mean and statistical significance (P < 0.05) is noted using *.